This proposal will determine new mechanisms governing the onset and progression of ErbB2-mediated breast tumorigenesis using inducible transgenic systems developed in the last funding period. Breast cancer is the second commonest cause of cancer death in women in the United States. ErbB2 overexpression and cyclin D1 overexpression is seen in 30%- to 50% of patient's tumors. ErbB2-targeted therapies with antibodies results in 2% complete and up to 12% partial response rates, suggesting opportunities exist to improve our current understanding of ErbB2-dependent tumorigenesis. Correlative studies show increased expression and/or activity of Akt/NFkB/Notch and cyclin D1 in both murine and human breast cancers. In the last funding period we established ErbB2-induced mammary tumorigenesis involves an Akt/NFkB/Notch1/cyclin D1 signaling pathway. The current proposal use tissue-specific ponasterone-inducible mammary gland targeted transgenic and knockout mice generated during the last funding period to determine the mechanisms by which ErbB2 governs induction vs. maintenance of mammary tumorigenesis. Aim 1. Determine the mechanisms by which Akt1 governs ErbB2-dependent mammary tumor progression. By crossing Akt1-/- and MMTV-ErbB2 transgenics we demonstrated that Akt1 contributes to ErbB2-induced tumorigenesis. We have identified a novel heterotypic signaling pathway by which Akt1 drives cellular invasion and migration. Using these multigenic mice we will define the mechanisms by which Akt1 regulates ErbB2-induced growth, migration and invasion. Aim 2. Determine the mechanisms by which Notch1 governs ErbB2-dependent mammary tumor progression. Our prior studies identified a Notch1 pathway regulating tumor growth. We have generated transgenic mice that regulate temporal and spatial control of activated Notch1 targeted to the mammary gland (Inducible Notch1IC) and developed mice in which endogenous Notch1 can be deleted from the mammary gland (Notch1fl/fl, MMTV-Cre). These transgenic mice will be used to determine the role of activated and endogenous Notch1 in ErbB2-mediated mammary gland tumorigenesis in vivo. Aim 3. Determine in vivo significance of NFkB/cyclin D1 in ErbB2-dependent mammary tumor progression. Deletion of cyclin D1 or NFkB in the mouse abrogates normal development of a mammary epithelial cell population that may contribute to tumorigenesis. We developed transgenic mice expressing an inducible mammary gland-targeted inhibitor of NFkB (IkBaSR), and inducible mammary gland-targeted cyclin D1 anti-sense transgenic mice. We have shown that NFkB is required for the onset and progression of ErbB2-mediated mammary tumorigenesis. These transgenic mice will be used to determine the mechanism by which NFkB and cyclin D1 contribute to the onset and progression of ErbB2-mediated mammary tumorigenesis. A select subset of experiments will be conducted using each of these unique transgenic mice (Aims 1-3) to generate a unique understanding of the role of miRNA and stem cell precursors in ErbB2-induced tumorigenesis. Together these three integrated Aims will determine the key events governing ErbB2-mediated tumor onset in vivo providing the basis for a rational approach to new therapies for this disease.